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“Without 3D information it is very difficult to understand how the genome works”

Marc A. Marti-Renom is interested in three-dimensional structures. After eight years in the US dedicated to the world of proteins, the biophysicist returned to his native country, first Valencia and then Barcelona, to specialise in RNA and DNA folding. In 2006 he set up his own group, which today is divided between the CNAG, where there are ten people, and the CRG, where there are two. “We do the experimental part, the sample preparation, here in the CRG, and the sequencing and analysis happens in the CNAG”, he explains. For his research he requires a large sequencing and computing capacity, which he can get at the CNAG, the second-most important sequencing analysis centre in Europe. “We are fortunate to be in one of the best places in the world to do these studies,” he says proudly.

3, MARC MARTI RENOM_14_group

 

Proteins with clinical application

Proteins caught his attention while he was doing his PhD, and in 2004, when he was at the University of California (UCSF), he collaborated in the creation of the “Tropical Disease Initiative,” a drug-discovery initiative linking people from both academia and companies to try to reposition drugs in favour of neglected diseases such as malaria and tuberculosis. “The idea was to make it all open source so everything we found was published directly to the web and couldn’t be patented”, says Marti-Renom.

The Structural Genomics group was a major player in one of the first instances that genome sequencing was used at the clinical level. “There was a patient with tuberculosis and a high resistance to antibiotics. We sequenced samples from the patient and found out he was infected by two different strains, and one of them was mutated. When we made models of the protein structure resulting from this mutation we saw how it was affecting the function”, explains the scientist. According to Marti-Renom, in a few years not only will everyone have their genome sequenced, but it will happen several times. “When someone develops a disease like cancer we will sequence them again to see what has changed and why”, he predicts.

Beyond proteins: RNA and DNA

Proteins, the cell’s building blocks, are not the be-all and end-all of life. Since the 1960s we have known that RNA has essential functions other than converting the information in DNA into proteins. But of its three-dimensional structure very little is known, and in the end, the function occurs in 3D. For this reason the group is developing computational tools to incorporate experimental data and make structural predictions.

The most recent biological component to enter the ‘3D world’ was the genome. In this case, too, little is known about how it folds in space. The group of Marti-Renom, along with three other groups at the CRG (Miguel Beato, Guillaume Fillion and Thomas Graf) is carrying out the 4DGenome project, which has a budget of 12.2 million euros, in order to understand the structure of the genome and how it changes over time. “We know the genome sequence very well, thanks to molecular biology and the big genome projects. We also understand the chromosomal macrostructure, thanks to advances in microscopy; but we can’t see the middle ground, the step between the tangled skein and the well-defined chromosome”, says the head of the group. In 2006 they began using Chromosome Conformation Capture (3C) data to develop software that allows you to view the entire genome at high-resolution, a kind of ‘molecular microscope’. With this and other technologies, like Hi-C, and using computational algorithms they have been able to observe how different regions of the same chromosome tend to interact with each other. They have also seen that the 3D ‘photo’ of a moment when, for example, there is high gene expression may be very different to another where the expression is low. “Without this three-dimensional information it is much more difficult to characterise how the genome works”, concludes the researcher.

This article was published by Maruxa Martinez, Scientific Editor, at the El·lipse publication of the PRBB.

“We’re evolving towards systems pharmacology”

 

Jordi Mestres in the lab

A theoretical chemist by training, Jordi Mestres started up the chemogenomics lab of the IMIM, currently part of the GRIB, in 2003. The structure of the group, made up of graduates and doctors in chemistry, biology, biotechnology and computer science, perfectly reflects its three main lines of research: molecules, proteins and programming to predict the interaction between them.

“We apply our predictions to both drug discovery and chemical biology”, summarises Mestres. This last discipline consists of using small molecules to sound out biology, for example inhibiting a protein to understand its function. According to the scientist from Girona the optimisation of these chemical probes is just as important as that for drugs. “They have been used for years as if they were selective for a single target protein, but now we are beginning to understand that they are not.”

In fact, drugs do not owe their effectiveness to the fact that they are very selective for a single target, rather to their affinity for a whole group of proteins. “We are evolving towards systems pharmacology, where the drug is placed in the context of all of the proteins with which it can potentially interact, the organs it can reach, the polymorphisms of the person that takes the drug, and so on”, explains the head of the group.

 

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 A multitude of projects

The laboratory is involved in several European projects, including Open PHACTS, where they have developed an interactive tool to show ligand-protein interactions via the web (www.pharmatrek.org), and eTOX coordinated by Ferran Sanz (GRIB), where they design new methods to predict drug safety profiles. “Drug safety profiles are not really known until they are on sale and the drug is exposed to millions of users. If we were able to anticipate any adverse effects before entering the market and we understood the mechanisms, we could modify the structure of the drug in advance”, reasons Mestres.

They also look at ethnopharmacology, and try to explain how medicinal plants work. “We have made predictions for 109 plants and we are trying to rationalise their use for cardiovascular disease.”

In collaboration with Pilar Navarro (IMIM) they have found molecules inhibiting the formation of b-amyloid plaques that work as well or better than memantine, an Alzheimer’s drug. The research was funded by a pharmaceutical company and has generated two patents. In total, the group has four patents in collaboration with companies and one with the CSIC.

The creation of a spin-off

In some cases, they are asked by companies or other groups to prioritise which molecules to use at the beginning of a research project or to predict the proteins of active molecules in phenotypic trials. This was the origin of Chemotargets, in 2006, where currently three people work. “The students who were doing this could not publish anything, so we created this spin-off service”, explains the head of the group.

Chemotargets is still going and has quite a lot of work. They are currently designing the screening collection for the Karolinska Institute in Stockholm, with more than 10,000 molecules. They did something similar for the CRG, creating a list of small molecules that interact with proteins of interest to the researchers. Lately, they have also been contracted by the Swiss foundation ‘Medicines for Malaria Venture’ (MMV) to investigate the action of 400 antimalarials identified in phenotypic tests. “Chemotargets predicts targets for each molecule. Afterwards it is necessary to confirm the predictions experimentally, and this work is usually outsourced”.

It is, according to Mestres, the future of drug design. “Everything will be done from an office in a skyscraper in Manhattan or London, outsourcing molecule design to companies like Chemotargets, synthesis to a chemical company in China, and the trial to a pharmacology firm in India”, he predicts. “In fact it is already happening with the big pharmaceutical companies -they close their research centres, but do not abandon projects: they subcontract them out”.

 

Pedro Carvalho (CRG) explains the role of ER in protein control and lipid homeostasis

Pedro Carvalho, from the CRG tries to understand how the cell uses its quality control mechanisms to get rid of proteins that are not functional. He also studies lipid homeostasis. Both functions take place at the Endoplasmic Reticulum (ER). Check out this video to hear him explain his research.

“The same mutation can have a different outcome in different individuals”

The English researcher Ben Lehner started as a junior group leader at the CRG in December 2006 and has been an ICREA Professor since 2009. His lab, Genetics Systems, consists of five postdoctoral fellows, four PhD students, and a technician who hail from Italy, the UK, Germany, the Netherlands, Poland, Chile, Peru, Canada and Switzerland. About half of the group members are computational biologists and the rest work primarily in the ‘wet’ lab. They all have the same aim – to understand basic questions in genetics – but they use diverse approaches and model systems.

From individual genome sequences to individual phenotypes 

“In humans many mutations in genes are associated with an increased risk of particular diseases such as cancer”, the scientist explains. “But human geneticists are terrible at predicting disease risk. Most people with disease mutations never get the disease”. One aim of Lehner’s group is to better understand how the thousands of mutations in the genome of a particular individual interact to influence phenotypes such as disease risk. “What causes the same mutation to have a different outcome in different individuals? That’s one of our favourite questions”, states the researcher. This means understanding how genetics, the environment and ‘chance’ influence the outcome of particular genetic variants.
Recent research has focused on finding ways to predict the ‘normal’ outcome when genes are inhibited. In collaboration with labs from Korea, Toronto and Texas the group has created prediction models using generalisations such as the shared function of genes. “If two proteins interact physically, one can assume that they are involved in similar processes”, Lehner explains. The consequence of this hypothesis is that a mutation in either of the genes is likely to result in a similar phenotype, according to their shared function. By assuming this, one can generalise and expand the findings by using all available information on physical data, genetic interaction and co-evolution of every single gene analysed.

The next step for the group is to understand how the thousands of mutations in an individual’s genome combine to influence their characteristics. “Two humans differ by thousands of mutations, so how do we evaluate the outcome of all of this genetic variation in one go?” Beyond this they are also trying to understand why it can be impossible to predict disease phenotypes from a genome sequence. “Even genetically ‘identical’ twins are not identical when it comes to disease susceptibility. The same is true in simple organisms – if you control the genetics and you control the environment, you still cannot predict what will happen. We are working to understand why this is”, says the head of the lab.

Focus on basic problems 

In their studies the group uses experimentally tractable model organisms like yeast or the transparent roundworm C. elegans, but they also use existing data from many different sources. “Rather than working with a single system or approach, we like to choose the best system to study a particular problem with. And if you look at the history of biology, ‘best’ normally means ‘simplest’”, explains the biologist. “The problem is the important thing – it doesn’t really matter how you choose to solve it. But the problem must be basic and general, and one that you can actually solve!”.

This article was published in the El·lipse publication of the PRBB.

Andreas Meyerhans

Andreas Meyerhans, from the Department of experimental and health sciences at the Pompeu Fabra University (CEXS-UPF), located at the PRBB,  explains in one minute his group’s research. They study acute and persistent infections, and try to find out what makes an infection become one or another.

Notch in normal and leukemic cells

Anna Bigas and Lluís Espinosa, of the Stem Cell and Cancer group of the IMIM are two principal investigators who have joined forces to investigate different aspects of cancer development. Together with their jointed group of 14 researchers, Bigas focuses on hematopoietic stem cells, while Espinosa concentrates on solid cancer and intestinal stem cells.

Bigas aims to understand how a pluripotent stem cell becomes a hematopoietic stem cell during embryogenesis. `It is a great challenge in the regenerative medicine field to understand where these stem cells come from and how they conserve this self- renewing capacity which enables them to maintain a tissue´, she explains. She focuses on a major signalling pathway controlling decisions in both normal and leukemic cells and which is also important for tissue maintenance: the Notch pathway.

Searching for Notch target genes

In order to specify the molecular mechanisms driving an undifferentiated cell towards the hematopoietic lineage or to the leukemic phenotype, the group’s current objective is to find Notch target genes and to describe their mechanism of function.

The researchers use techniques like such as chromatin precipitation and promoter arrays. This results in lists of possible candidate genes from which the real targets have to be isolated and validated in a series of experiments, including FACS to isolate the cells of interest and to determine whether the candidate gene is expressed. Further molecular and biochemical analysis, as well as experiments using mutant mice, help define possible interactions of the target molecules and their effect in the organism.

One target gene is GATA2, an important hematopoietic transcription factor that is not expressed in Notch mutant mice and is altered in human leukaemia. The Bigas group have characterized the GATA2 promoter and found that Notch exerts both positive and negative signals that restrict the intensity and the duration of GATA2 expression in hematopoietic cells.

In parallel, Espinosa is studying whether Notch cooperates with other signalling pathways in different contexts. He found that specific elements of the NF-κB pathway, which is involved in cancer development, directly regulate the transcription of genes which are known to be Notch-dependent.

In a common work Bigas and Espinosa identified a new role of the Notch signalling pathway in the maintenance of leukemic stem cells. Previous studies had shown that both the NF-κB and the Notch pathway are involved in T-cell acute lymphoblastic leukaemia, and future therapeutical strategies may employ both Notch and NF-kB inhibitors to fight this leukaemia.

However only the recent findings of Bigas and Espinosa describe the exact mechanism by which Notch activates the NF-kB transcription factor. These new insights published in Cancer Cell, could be translated to clinical trials and result in better pharmaceutical treatments with less side effects.

Despite all the advances in the field, it is not yet known whether Notch is also important for the leukaemia initiating cells, a question that Bigas would like to answer in the close near future. These cells are of major interest, since they are resistant to standard leukaemia treatments, remain in the organism and ultimately are the source of a recurrent outbreak.

Bigas and Espinosa maintain collaborations both inside and outside of the PRBB. `It is a great advantage to have so many scientists within a few square metreers´ Bigas states, `and we just have a great collaborative work in progress, including involving scientists from the CRG, the UPF, Hospital del Mar and others´.

This article was published in the El·lipse publication of the PRBB.

“We are the interface between industry and academia” – Computer-assisted drug design lab

The Computer-Assisted Drug Design (CADD) laboratory of the GRIB is devoted to the area of drug design and development. Directed by Manuel Pastor, who started the group 10 years ago at the IMIM, it includes pharmacists, biologists, chemists, and a mathematician. “We also had a telecommunications engineer at one point. Our research needs experts in both science and programming”, justifies Pastor.

The group’s interests are divided into three main areas. The first is methodological: they have written several programs marketed and are used by many pharmaceutical companies. The most recent one is Pentacle, which allows the creation of models relating the structure with the activity of a compound, as well as the computation of molecular descriptors. “Molecular descriptors are used to convert a real molecule into a computer representation, so they are needed in pretty much all steps of drug development”, explains the head of the group. Another software tool created by the group a few years ago is Shop. “Imagine you have a molecule that has the desired effect, but it cannot be used: because, for example, it’s toxic, or not soluble enough. With Shop you can remove the fragment that causes the problem and substitute it for another that will maintain the same biological activity without the side effect”.

A second research area is structure-based drug design (SBDD), which they apply to the study of schizophrenia. In collaboration with other groups in Santiago de Compostela they are looking at potential drug targets for the disease to try to find new compounds that can bind them.

Innovating Medicines 

The most recent and active research area is focused on drug safety. The group coordinates the IMI (Innovative Medicines Initiative) project eTOX, which aims to develop methodologies to predict toxic properties of new compounds in silico (with the help of computers) and as early as possible. “This project involves pretty much all the big pharmaceutical companies in Europe, as well as some of the best European academic groups in cheminformatics”- says Pastor – “Pharma companies have realised that what is making it difficult for them is not the competition, but the intrinsic complexity of the problem. They have run out of easy targets, and the existing methodologies are not working that well finding good drugs for the difficult ones. So they are starting to join forces”.

Another upcoming IMI project Pastor is excited about is OpenPhacts (Open Pharmacological Space). As with eTox, Pastor will collaborate with Ferran Sanz and other GRIB members, to contribute to this project which “will change the scene completely”, promises the head of CADD. “There is a lot of information publically available information that is of interest to pharmaceutical companies: data on compounds, structures and, pathways. But it’´s all dispersed, and the industry is spending huge amounts of money trying to collect and exploit this information. OpenPhacts will bring together make pertinent enquiries, such as: is there a compound similar to this one involved in this specific pathway? The CADD group will contribute by assessing these relevant questions. “The drug companies know what they need, and the technical experts know what can be done. We are the intermediaries, we know about both worlds”, concludes Pastor.

This article was published in the El·lipse publication of the PRBB.

Listening to the language of neurones

Coming from the Rockefeller University in NY, Matthieu Louis leads the Sensory Systems and Behaviour group at the CRG, the only lab in Barcelona, and one of the few in Spain, investigating Drosophila neuroscience. His team comprises eight people with backgrounds in molecular biology, engineering and physics. Their aim is to correlate neural circuit function with behaviour using fruit fly larvae. “The Drosophila larva has a repertoire of complex behaviours and key cognitive functions. Yet its nervous system has 10 million neurones fewer than humans”, explains the physicist.

The group tries to understand how odours are encoded by the olfactory system. Features such as quality, “Does this smell like banana?”, and intensity, “Is this a morsel of banana or a bunch?”, are efficiently represented by only 21 olfactory sensory neurones, so that the larva can distinguish between hundreds of food-related odours. The researcher says that there must be a combinatorial code, yet it does not seem to be as trivial as the activation of different combinations of neurones by distinct odours. “We have evidence that the nature and the intensity of an odour is represented not only by the identity of the sensory neurones it activates, but also the way each one is activated”, he explains.

From information processing to chemotaxis 

Once a smell has been encoded, it has to be processed. To find the higher-order neurones involved in the integration of olfactory information, the group is undertaking a large behavioural screen. They test thousands of fly lines in which subsets of neurones are inhibited or over-activated. They then characterise how these perturbations affect chemotaxis, the orientation behaviour observed in response to an odour gradient. To decide whether to go straight ahead or turn, the larva monitors information about odour concentration changes. When a wild-type animal detects an intensity increase of an attractive odour, it keeps going forwards, but, as the group has recently described, if the odour intensity decreases the larva reorients through an active-sampling mechanism: much like rats and dogs, the larva sweeps its head laterally to check intensity levels on either side.

Drosophila larva

With their screen, the researchers are looking for mutants showing reorientation defects. To that end, they have developed their own computer-vision software. “We needed an algorithm to quantify subtle movements of the head and body posture with a high space-time accuracy. As no tool like this existed, we spent a year developing one”, says the head of the group.

Predicting behaviour 

“If we understand the neural logic of larval chemotaxis well enough, we should be able to synthetically produce predictable behavioural sequences”. Such a model could be useful for robotics. “Currently, dogs are trained to find mines. We could design robots that navigate spatially, searching for the chemical compounds present in explosives”.

Many questions remain to be answered before this becomes reality. How does the larva integrate a series of stimuli (touch, light, heat, smell) that are received simultaneously before deciding what to do next? And how is sensory input converted into motor output? “There are many challenges ahead. But it is thrilling to witness the genesis of a decision in a minibrain, from elementary spikes in sensory neurones down to the coordinated contraction of dozen of muscles. Flies have much to teach us about the function of our own brain”, concludes the Belgian researcher.

This article was published in the El·lipse publication of the PRBB.

Studying how evolution has shaped our genome- Arcadi Navarro (UPF) explains

In this video you can hear Arcadi Navarro, from the Institute for Evolutionary Biology (IBE), a mixed centre from the CSIC and the Pompeu Fabra University (UPF). His group studies how evolution has shaped our genomes and those from other species.

Doing real translational research: from patient to molecule

The Myogenesis, Inflammation and Muscle Function group from IMIM-Hospital del Mar Research Institute, coordinated by Joaquim Gea, is one of the few groups where a real translational research is already taking place. Formed 15 years ago by a group of clinical doctors specialized in respiratory diseases and a group of more basic biologists, its aim is to find direct links between specific symptoms and cellular and molecular mechanisms. For this, they do a clinical evaluation of the patient (or the animal model), obtain biological samples and do physiological studies. This part is done at the human physiology laboratory at the Hospital del Mar (or at the PRBB animal facility if it is an animal model). Then, at the respiratory biology lab at IMIM, the cellular and molecular analysis of the samples takes place.

Research at the unit is aimed at the diagnostic and treatment of respiratory diseases such as lung cancer, the cause of 20% of all cancer deaths. They also study chronic obstructive pulmonary disease (COPD), a disease caused by smoking which is more and more frequent (9% of the adult population, or 14% in men only). Sepsis (an extended blood infection), the most frequent cause of death at intensive care units, which causes exaggerated inflammatory mechanisms all through the body, is another of the diseases studied here. The group has also small projects in other non-respiratory disease, such as adolescent scoliosis (an anomalous rotation of the spinal cord) and fibromialgy, a disease which causes strong pains and tiredness and which affects 2-5% of the adult population, especially women.

The group is internationally-recognized and the most outstanding Spanish laboratory in the respiratory muscles field. It is divided into three research lines which cover different pathological mechanisms of their diseases of interest: local inflammation as a signal mechanism, a line directed by Joaquim Gea; structural and immunological alteration of the muscles and the respiratory tracks, directed by Mauricio Orozco-Levi; and local and systemic oxidative stress, directed by Esther Barreiro, the only one who is exclusively involved with research and is, for the moment, not doing any clinical activity. The three of them combine research with teaching at the UPF.

According to Gea, the biggest challenge in this field is to find drugs for the specific targets that are being identified. Interestingly, the mechanisms of loss of muscular function are very similar for the different diseases, so some of the common symptoms could have similar therapeutic approaches.

Apart from their own studies, the unit also acts as a reference laboratory, designing studies for other groups and analyzing samples from different regions in Spain, the UK, The Netherlands and Canada. The unit also collaborates with other groups at the PRBB, such as that of Pura Muñoz (CEXS-UPF) and that of Josep M. Antó (CREALIMIM).

This article was published in the El·lipse publication of the PRBB.

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